Development of high-functional production processes using microbial cyclic-amide metabolism

开发了利用微生物环酰胺代谢来生产有用化合物的新工艺。一株同化丁二酰亚胺的细菌恶臭假单胞菌S52被发现能有效地从富马酸中产生丙酮酸。在富马酸浓度为2%(w/v)的条件下，发酵27h可使500 mM富马酸转化为286 mM丙酮酸，恶臭假单胞菌s52获得了溴丙酮酸抗性突变株，并对其丙酮酸产量进行了测定。其中一株突变株15160的丙酮酸产量远高于亲本菌株。以富马酸浓度为2%(w/v)的突变细胞为催化剂，用1000 mM富马酸在72 h内合成770 mM丙酮酸，发现亚麻短杆菌C-L产生了一种新型的环酰胺水解酶，该酶能立体选择性地降解5-取代的噻唑烷二酮，并可用于生产光学活性的α-巯基酸。从甜菜夜蛾品系C-1的染色体DNA中克隆了该酶的编码基因，并进行了序列测定。它包含一个开放阅读框，由936个核苷酸组成，对应312个氨基酸残基。推导的氨基酸序列与蛋白质数据库中的其他蛋白质序列没有明显的同源性。该酶在重组大肠杆菌中得到了表达。转化大肠杆菌BL21/pETBTH是以RS-5-苄基噻唑二酮为原料立体选择性合成S-3-苯基-2-硫代丙酸(S-PMPA)的良好催化剂。制备的S-PMPA的光学纯度约为80%e.E.

Novel processes for the production of useful compounds using microbial cyclic-amide metabolism were developed. A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using the cells cultivated in the medium containing 2% (w/v) fumarate as the catalysts, 286 mM pyruvate was produced from 500 mM fumarate in 27 h. Bromopyruvate-resistant mutants were derived from P. putida s52, and their pyruvate production was examined. One of the mutants, strain No. 15160 showed much higher pyruvate production than the parent strain. Using the mutant cells cultivated in the medium containing 2% (w/v) fumarate as the catalysts, 770 mM pyruvate was produced from 1000 mM fumrate in 72 h. Brevibacterium linens C-l was found to produce a novel cyclic amidohydrolase which stereoselectively hydrolyzes 5-substituted thiazolidinedione and wasuseful for the production of optically active α-mercapto acids. The gene encoding the enzyme was cloned from the chromosomal DNA of B. lines C-1 and sequenced. It contains an open reading frame consisting of 936 nucleotides corresponding to 312 amino acid residues. The deduced amino acid sequence exhibited no apparent homology with other protein sequences in the protein databases. The enzyme was expressed in recombinant E. coli cells. The E. coli transformant, E. coli BL21/pETBTH, was a good catalyst for stereo selective production of .S-3-phenyl-2-mercaptopropionic (S-PMPA) from RS-5-Benzyl-thiazolidinedione. The optical purity of S-PMPA produced was approximately 80% e.e.